Studies of the developing dental pulp innervation by nerve fibers from the trigeminal ganglion is an excellent example of target tissue/neuron interactions and will allow specific questions regarding the development of the pulpal nervous system to be addressed. The dental pulp is a unique model system in which to study target tissue-nerve interactions. It is anatomically well-defined and becomes richly innervated by trigeminal nerve fibers. Dental pulp innervation occurs at a rather late postnatal age compared to other trigeminal targets after the basic structure of the tooth organ has been already formed. It is easily accessible at early postnatal ages and can be isolated for in vitro studies. Dental pulp cells produce an array of potent neurotrophic factors during development. We have established an in vitro model to grow co-cultures of dissociated dental pulp cells and trigeminal neurons and study their interactions in vitro. This in vitro system is the first single cell neuronal co-culture model to be established without the addition of exogenous neurotrophic factors. Neurons in our system are not selected by exogenous neurotrophic factors that are added into the culture system, neuron survival is rather determined by dental pulp-derived neurotrophic factors, resembling the in vivo situation. We have also developed and refined an intraocular transplantation technique to study innervation of the dental pulp in oculo. This technique provides us with an excellent tool to study how different neurotrophic factors influence the innervation of the dental pulp. With these models, I propose to study the role of neurotrophic factors in establishing the innervation of the dental pulp. The most important initial studies aim to characterize the different cell types in our culture system and determine which neurotrophic factors are produced by dental pulp cells in culture. Using different markers for pulpal cells, immunohistochemistry and in situ hybridization will be applied separately and/or in combination to characterize different cell types. RNase protection assays in combination with the above-mentioned techniques will be used to characterize which neurotrophic factors are produced and which cell type is the source for the production of a specific neurotrophic factor. The proposed studies will enable us to characterize different cell types in our culture system, provide us with significant data regarding if and how neurotrophic factors influence the pattern of tooth innervation and will serve as basis for a future Individual Research Proposal (R01). The long-term goal of this laboratory is to understand the molecular mechanisms that underlie dental pulp innervation, based on which, we aim to develop therapeutical strategies for diseases in, and injuries of, the nervous system. Our preliminary data indicate that dental pulp cells provide neurotrophic support for many different types of neurons in vitro including the dopaminergic neurons of the ventral mesencephalon. These neurons are lost in Parkinson's disease. One of our future goals is to develop a transplantation therapy for neurodegenerative diseases based on the use of dental pulp cells, therefore it is of outmost importance to characterize different cell types and neurotrophic factors in the dental pulp cultures.